The purpose of this grant is to generate a novel series of agents for genetic manipulation of receptors, ion channel, and signaling molecules in vivo. These agents (tethered toxins) are chimeric molecules derived from tethering of naturally occurring peptide neurotoxins to the cell surface via GPI anchors or transmembrane. These studies derive from the discovery of mammalian prototoxin genes (e.g. Lynx 1) which are the evolutionary antecedents of snake venom toxins, and which can function as modulators of nAChRs in their native GPI-anchored form. Preliminary results are that tethered bungarotoxins retain their activity on nAChRs, and that they are not cleaved from the cell surface to inhibit adjacent cells. The existence of many thousands of naturally occurring peptide neurotoxins (e.g. bungarotoxins, conotoxins, conantokins, etc.), their exquisite target specificities, and the ability to target expression of the agents in vivo using BAC transgenic mice, suggests that the development of a generic strategy for harnessing their potency for in vivo use will permit genetic control over a wide variety of neuronal functions. For example, cell specific genetic control of neural activity, neurotransmitter receptor function (e.g. ACh, NMDA, 5-HT3 receptors), and specific GPCR signal transduction cascades would become possible. The specific aims are to: 1) Construct additional tethered toxins, particularly tethered conotoxins, and test their activity in Xenopus oocytes; 2) Produce BAC transgenic mice expressing tethered toxins in specific CNS cell types in vivo. Assess the efficacy of tethered toxin action by evaluating phenotypes that would be expected based on results obtained in Specific Aim 1 and current knowledge of the roles of the targeted receptors and ion channels in vivo; 3) Develop inducible tethered toxins to improve the temporal resolution of this strategy for genetic manipulation of specific cells and signaling pathways. These studies will allow unprecedented precision in the genetic dissection of functions required for CNS development, function and dysfunction in vivo.